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  • Title: Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.
    Author: Ruvinov SB, Thompson J, Sackett DL, Ginsburg A.
    Journal: Arch Biochem Biophys; 1999 Nov 01; 371(1):115-23. PubMed ID: 10525296.
    Abstract:
    Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at </=15 degrees C and yields of active tetramer increased as the concentration of unfolded subunits decreased. Refolding of unfolded subunits and active tetramer assembly upon 100-fold dilution from 5 M GdnHCl at 0 degrees C also was increased two- or fourfold (to 44 or 28% reactivation for 0.08 or 0.28 microM subunit, respectively) when incubated at 15 degrees C, pH 7.2, for 4 h with the Escherichia coli molecular chaperonin GroEL, ATP, MgCl(2), and KCl.
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